A gene for achondroplasia-hypochondroplasia maps to chromosome 4p.

Achondroplasia (ACH) is a frequent condition of unknown origin characterized by short-limbed dwarfism and macrocephaly. Milder forms, termed hypochondroplasias (HCH) result in short stature with radiological features similar to those observed in ACH. We report on the mapping of a gene causing ACH/HCH to human chromosome 4p16.3, by linkage to the iduronidase A (IDUA) locus, in 15 informative families (Z max = 3.01 at theta = 0 for ACH; Z max = 4.71 at theta = 0 for ACH/HCH). Multipoint linkage analysis provides evidence for mapping the disease locus telomeric to D4S412 (location score in log 10 = 4.60). Moreover, this study supports the view that ACH and HCH are genetically homogeneous in our series.

Mutations of the TWIST gene in the Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome (acrocephalo-syndactyly type III, ACS III) is an autosomal dominant craniosynostosis with brachydactyly, soft tissue syndactyly and facial dysmorphism including ptosis, facial asymmetry and prominent ear crura. ACS III has been mapped to chromosome 7p21-22. Of interest, TWIST, the human counterpart of the murine Twist gene, has been localized on chromosome 7p21 as well. The Twist gene product is a transcription factor containing a basic helix-loop-helix (b-HLH) domain, required in head mesenchyme for cranial neural tube morphogenesis in mice. The co-localisation of ACS III and TWIST prompted us to screen ACS III patients for TWIST gene mutations especially as mice heterozygous for Twist null mutations displayed skull defects and duplication of hind leg digits. Here, we report 21-bp insertions and nonsense mutations of the TWIST gene (S127X, E130X) in seven ACS III probands and describe impairment of head mesenchyme induction by TWIST as a novel pathophysiological mechanism in human craniosynostoses.

Bypassing melanocyte senescence by beta-catenin: a novel way to promote melanoma.

The Wnt/beta-catenin signaling pathway plays a key role in several cellular functions during embryonic development and adult homeostasis. The deregulation of this pathway may lead to the development of cancer, including melanoma. Deregulation of the Wnt/beta-catenin pathway occurs through either the induction/repression of, or specific mutations in, various members of this signaling pathway; this results in the stabilization of beta-catenin and its translocation from the cytoplasm to the nucleus, where it regulates transcription. Although nuclear beta-catenin is clearly involved in malignant transformation, the mechanism by which it exerts its effects remains elusive. This review focuses on the molecular and cellular mechanisms that are driven by beta-catenin and lead to melanocyte transformation. In particular, we describe how beta-catenin induces melanocyte immortalization, a novel activity of this multifunction protein. Finally, we discuss how beta-catenin-induced immortalization can cooperate with MAPKinase pathways to produce melanoma.

Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia.

We report the absence of functional parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors (PTH/PTHrP receptor) in Blomstrand chondrodysplasia, a genetic disorder characterized by advanced endochondral bone maturation. Analysis of PTH/PTHrP receptor genomic DNA from a patient with Blomstrand chondrodysplasia demonstrated that the patient was heterozygous for a point mutation (G--> A substitution at nucleotide 1176) inherited from the mother. Analysis of PTH/PTHrP receptor cDNA demonstrated that: (a) this point mutation caused the deletion of the first 11 amino acids of exon M5 (encoding the fifth transmembrane domain of the receptor), resulting from the use of a novel splice site created by the base substitution; (b) the mutant receptor was well expressed in COS-7 cells, but did not bind PTH or PTHrP, and failed to induce detectable stimulation of either cAMP or inositol phosphate production in response to these ligands; and (c) the paternal allele was not expressed. Thus, only the abnormal and nonfunctional PTH/PTHrP receptors encoded by the maternal allele were expressed by chondrocytes from this patient. In view of the known role played by the PTH/PTHrP receptor in bone and cartilage development, these results strongly support the conclusion that the absence of functional PTH/ PTHrP receptors is responsible for the skeletal abnormalities seen in Blomstrand chondrodysplasia, abnormalities that are the mirror image of those observed in Jansen's chondrodysplasia. These findings emphasize the importance of signaling through this receptor in human fetal skeletal development.

Abnormal procollagen synthesis in fibroblasts from three patients of the same family with a severe form of osteogenesis imperfecta (type III).

Dermal fibroblast cultures from three siblings with a severe form of osteogenesis imperfecta were established in order to analyze their procollagen and collagen synthesis. Cell strains from clinically normal consanguineous parents (first cousins), were also obtained for comparison. Total collagen production in culture media was diminished by 55% in the patients fibroblasts and to a lesser extent in the parents. This decrease was specific for collagenous proteins. From polyacrylamide gel electrophoresis, it appeared that the three children had not only the same defective secretion of pro alpha 1(I) molecules but that their pro alpha 1(I) migrated slightly faster than the parental and control counterparts. Analysis of secretion confirmed a reduced rate in procollagen synthesis and the absence of intracellular storage. Upon pepsin treatment, extracellular alpha 1(I) and alpha 2(I) chains were found in the expected ratio of 2:1 and migrated normally, suggesting that the altered mobility of pro alpha 1(I) chains was related to COOH or NH2 terminal propeptides. In agreement with the reduced type I collagen production, an increase in the alpha 1(III)/alpha 1(I) ratio was also detected. Furthermore, after a 2.5-h labelling followed by alkylation with iodoacetamide, free intracellular pro alpha 2(I) and alpha 1(I) chains were detected in the absence of reduction, consistent with an abnormal intracellular ratio of pro alpha 1(I)/pro alpha 2(I) that was measured after dithiothreitol reduction. Analysis of intracellular collagen chains from parental strains following a 4-h incubation demonstrated that pro alpha 1(I) appeared as a doublet, one band with normal mobility and a less intense band migrating faster and corresponding to the defective chain found in the patients. Absence of the abnormal molecules in culture media was related to the demonstration of a defective collagen secretion by parental fibroblasts. Correlation between these biochemical findings and clinical data strongly support a recessive inheritance of the disease that could be classified as a type III form of osteogenesis imperfecta. Patients would be homozygous for the same defective allele and the asymptomatic parents would most likely be heterozygous carriers of the mutation. Although the exact location of the alteration is not yet elucidated, a splicing mutation is suggested.

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes.

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.

Spatio-temporal expression of FGFR 1, 2 and 3 genes during human embryo-fetal ossification.

Mutations in FGFR 1-3 genes account for various human craniosynostosis syndromes, while dwarfism syndromes have been ascribed exclusively to FGFR 3 mutations. However, the exact role of FGFR 1-3 genes in human skeletal development is not understood. Here we describe the expression pattern of FGFR 1-3 genes during human embryonic and fetal endochondral and membranous ossification. In the limb bud, FGFR 1 and FGFR 2 are initially expressed in the mesenchyme and in epidermal cells, respectively, but FGFR 3 is undetectable. At later stages, FGFR 2 appears as the first marker of prechondrogenic condensations. In the growing long bones, FGFR 1 and FGFR 2 transcripts are restricted to the perichondrium and periosteum, while FGFR 3 is mainly expressed in mature chondrocytes of the cartilage growth plate. Marked FGFR 2 expression is also observed in the periarticular cartilage. Finally, membranous ossification of the skull vault is characterized by co-expression of the FGFR 1-3 genes in preosteoblasts and osteoblasts. In summary, the simultaneous expression of FGFR 1-3 genes in cranial sutures might explain their involvement in craniosynostosis syndromes, whereas the specific expression of FGFR 3 in chondrocytes does correlate with the involvement of FGFR 3 mutations in inherited defective growth of human long bones.

Genotype-phenotype correlation in hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a genetically heterogeneous autosomal dominant disorder characterised by the development of bony protuberances mainly located on the long bones. Three HME loci have been mapped to chromosomes 8q24 (EXT1), 11p11-13 (EXT2), and 19p (EXT3). The EXT1 and EXT2 genes encode glycosyltransferases involved in biosynthesis of heparan sulphate proteoglycans. Here we report on a clinical survey and mutation analysis of 42 HME French families and show that EXT1 and EXT2 accounted for more than 90% of HME cases in our series. Among them, 27/42 cases were accounted for by EXT1 (64%, four nonsense, 19 frameshift, three missense, and one splice site mutations) and 9/42 cases were accounted for by EXT2 (21%, four nonsense, two frameshift, two missense, and one splice site mutation). Overall, 31/36 mutations were expected to cause loss of protein function (86%). The most severe forms of the disease and malignant transformation of exostoses to chondrosarcomas were associated with EXT1 mutations. These findings provide the first genotype-phenotype correlation in HME and will, it is hoped, facilitate the clinical management of these patients.

EXT 1 gene mutation induces chondrocyte cytoskeletal abnormalities and defective collagen expression in the exostoses.

Hereditary multiple exostoses (HME), an autosomal skeletal disorder characterized by cartilage-capped excrescences, has been ascribed to mutations in EXT 1 and EXT 2, two tumor suppressor-related genes encoding glycosyltransferases involved in the heparan sulfate proteoglycan (HSPG) biosynthesis. Taking advantage of the availability of three different exostoses from a patient with HME harboring a premature termination codon in the EXT 1 gene, morphological, immunologic, and biochemical analyses of the samples were carried out. The cartilaginous exostosis, when compared with control cartilage, exhibited alterations in the distribution and morphology of chondrocytes with abundant bundles of actin filaments indicative of cytoskeletal defects. Chondrocytes in the exostosis were surrounded by an extracellular matrix containing abnormally high amounts of collagen type X. The unexpected presence of collagen type I unevenly distributed in the cartilage matrix further suggested that some of the hypertrophic chondrocytes detected in the cartilaginous caps of the exostoses underwent accelerated differentiation. The two mineralized exostoses presented lamellar bone arrangement undergoing intense remodeling as evidenced by the presence of numerous reversal lines. The increased electrophoretic mobility of chondroitin sulfate and dermatan sulfate proteoglycans (PGs) extracted from the two bony exostoses was ascribed to an absence of the decorin core protein. Altogether, these data indicate that EXT mutations might induce a defective endochondral ossification process in exostoses by altering actin distribution and chondrocyte differentiation and by promoting primary calcification through decorin removal.

Altered collagen of human pathological fibroblasts impairs the synthesis of fibronectin.

Human fibroblasts with mutated type I collagen have marked defective adhesive capacities on exogenous type I collagen and exogenous fibronectin in comparison to normal fibroblasts. This defective cell adhesion could be partly explained by the decreased level of cell surface receptors of the beta 1-integrin family, i.e., the alpha 2 integrin subunit for type I collagen and the alpha 5 integrin subunit for fibronectin, observed in pathological fibroblasts. However, it appeared that the presence of altered collagen interfered both with fibronectin biosynthesis and with its surface expression. Using a binding assay on immobilized fibronectin, we demonstrated that the mutated collagen had a weaker binding to fibronectin. In addition, the pathological fibroblasts plated on a mixture of normal exogenous type I collagen and fibronectin exhibited the same maximal level of adhesion as control fibroblasts. These results indicate that fibroblasts with the mutated collagen exhibit a decreased binding to normal fibronectin, a modification of synthesis and surface expression of fibronectin, and, finally, altered adhesive capacities.

Mutations within or upstream of the basic helix-loop-helix domain of the TWIST gene are specific to Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome (ACS III) is an autosomal dominant craniosynostosis syndrome recently ascribed to mutations in the TWIST gene, a basic helix-loop-helix (b-HLH) transcription factor regulating head mesenchyme cell development during cranial neural tube formation in mouse. Studying a series of 22 unrelated ACS III patients, we have found TWIST mutations in 16/22 cases. Interestingly, these mutations consistently involved the b-HLH domain of the protein. Indeed, mutant genotypes included frameshift deletions/insertions, nonsense and missense mutations, either truncating or disrupting the b-HLH motif of the protein. This observation gives additional support to the view that most ACS III cases result from loss-of-function mutations at the TWIST locus. The P250R recurrent FGFR 3 mutation was found in 2/22 cases presenting mild clinical manifestations of the disease but 4/22 cases failed to harbour TWIST or FGFR 3 mutations. Clinical re-examination of patients carrying TWIST mutations failed to reveal correlations between the mutant genotype and severity of the phenotype. Finally, since no TWIST mutations were detected in 40 cases of isolated coronal craniosynostosis, the present study suggests that TWIST mutations are specific to Saethre-Chotzen syndrome.

Mutations in the basic domain and the loop-helix II junction of TWIST abolish DNA binding in Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome is an autosomal dominant skull disorder resulting from premature fusion of coronal sutures (craniosynostosis). It is caused by mutations in the TWIST gene encoding a basic Helix-Loop-Helix transcription factor. Here we report on the identification of a novel mutation affecting a highly conserved residue of the basic domain. Unlike nonsense and missense mutations lying within helices, this mutation does not affect protein stability or heterodimerisation of TWIST with its partner E12. However, it does abolish TWIST binding capacity to a target E-box as efficiently as two missense mutations in the loop-helix II junction. By contrast, elongation of the loop through a 7 amino acid insertion appears not to hamper binding to the DNA target. We conclude that loss of TWIST protein function in Saethre-Chotzen patients can occur at three different levels, namely protein stability, dimerisation, and DNA binding and that the loop-helix II junction is essential for effective protein-DNA interaction.

Overexpression of FGFR3, Stat1, Stat5 and p21Cip1 correlates with phenotypic severity and defective chondrocyte differentiation in FGFR3-related chondrodysplasias.

Achondroplasia (ACH) and thanatophoric dysplasia (TD) are human skeletal disorders of increasing severity accounted for by mutations in the fibroblast growth factor receptor 3 (FGFR3). Attempts to elucidate the molecular signaling pathways leading to these phenotypes through mouse model engineering have provided relevant information mostly in the postnatal period. The availability of a large series of human fetuses including 14 ACH and 26 TD enabled the consequences of FGFR3 mutations on endogenous receptor expression during the prenatal period to be assessed by analysis of primary cultured chondrocytes and cartilage growth plates. Overexpression and ligand-independent phosphorylation of the fully glycosylated isoform of FGFR3 were observed in ACH and TD cells. Immunohistochemical analysis of fetal growth plates showed a phenotype-related reduction of the collagen type X-positive hypertrophic zone. Abnormally high amounts of Stat1, Stat5 and p21Cip1 proteins were found in prehypertrophic-hypertrophic chondrocytes, the extent of overexpression being directly related to the severity of the disease. Double immunostaining procedures revealed an overlap of FGFR3 and Stat1 expression in the prehypertrophic-hypertrophic zone, suggesting that constitutive activation of the receptor accounts for Stat overexpression. By contrast, expression of Stat and p21Cip1 proteins in the proliferative zone differed only slightly from control cartilage and differences were restricted to the last arrays of proliferative cells. Our results indicate that FGFR3 mutations in the prenatal period upregulate FGFR3 and Stat-p21Cip1 expression, thus inducing premature exit of proliferative cells from the cell cycle and their differentiation into prehypertrophic chondrocytes. We conclude that defective differentiation of chondrocytes is the main cause of longitudinal bone growth retardation in FGFR3-related human chondrodysplasias.

Morphological and biochemical studies of a mouse mutant (fro/fro) with bone fragility.

The mutation fragilitas ossium (fro) was discovered in a random-bred stock of mice during an experiment aimed at detecting recessive lethal mutations after treatment of the postmeiotic germ cells of male mice with tris (1-aziridinyl)phosphine sulphide. The affected mice were moderately runted and had deformities in all four limbs. The radiological and histological findings indicate that the mutant is similar to human osteogenesis imperfecta. The ash content of long bones was lower in the mutant. A defect of type I collagen could not be detected. The electrophoretic patterns of alpha bands of type I and V collagen and CB derived peptides of type I collagen from bone and skin showed no abnormalities. The total collagen synthesis and secretion in cultures of dermal fibroblasts, as well as the gel electrophoresis of procollagen and collagen chains synthesized, and of their CB peptides, were the same as those found in the controls. The percentage of type I and type V collagen synthesized was similar; that of type III was lower in the mutants. Bone osteonectin was found to be decreased by 30% and bone sialoprotein by 5%. The mRNA level for osteonectin was decreased in the fibroblasts of the mutant by about 50%. Whether the defective expression of the osteonectin in fro/fro mice is due to a mutation in the gene itself or its regulatory site(s), or is secondary to other factors remains to be established. The fro/fro mouse may represent a model for some forms of human bone fragility without collagen abnormalities.

Radiological and histological variants of thanatophoric dysplasia are associated with common mutations in FGFR-3.

We describe two fetuses of the 21st week of gestation that share some macroscopic, radiologic, and histologic findings of thanatophoric dysplasia (TD), but also show distinct differences from the usual subtypes of TD. These differences mainly comprise the lack of facial abnormality, only mild reduction of chondrocyte proliferation and hypertrophy, and the lack of fibrous tissue interposition between cartilage and periosteal bone. Thus, these two cases may represent a distinct variant of thanatophoric dysplasia. The molecular analysis of the FGF-R-3 gene demonstrated in both cases mutations which were not significantly different from those of other cases of TD. Thus, the phenotypic modulation within the subtypes of TD may be influenced by additional and yet unknown factors.

Dyssegmental dysplasia with glaucoma.

We report on a "new" syndrome in 2 unrelated children with some manifestations of Kniest dysplasia and with spine abnormalities suggestive of dyssegmental dysplasia. Glaucoma with important ocular impairment was associated with severe dwarfism. No mutation of the COL2A1 gene was detected. The inheritance of this new type of skeletal dysplasia in unknown.

Type II collagen defect in two sibs with the Goldblatt syndrome, a chondrodysplasia with dentinogenesis imperfecta, and joint laxity.

We report on a syndrome of spondylo-epimetaphyseal dysplasia, dentinogenesis imperfecta, and ligamentous hyperextensibility in two sibs born to nonconsanguineous parents. This chondrodysplasia was characterized by severe shortness of stature and an osteoporosis without fractures. Electron microscopic examination of the cartilage documented large vacuoles of dilated rough endoplasmic reticulum within the cytoplasm of chondrocytes. Gel electrophoresis of pepsin-soluble collagen extracted from cartilage demonstrated the presence of type II collagen chains with an abnormal mobility. Prolyl and lysyl hydroxylations were slightly increased. The abnormal molecules melted at a higher temperature than the normal ones. CNBr peptide mapping of type II collagen showed an altered electrophoretic migration of peptides CB 11, CB 8, and CB 10,5 whereas CB 9,7 looked normal. In addition, two small non-collagenous proteins isolated from cartilage were not found in an age-matched control individual but were detected in a normal newborn infant. The quantitation of proline-labelled collagen synthesized by dermal fibroblasts demonstrated a 50% reduction of total collagen. This decrease essentially affected the amount of extracellular type I collagen, which was secreted less efficiently than in control cells. Nevertheless, type I collagen chains behaved normally on 5% polyacrylamide gels. The reduced mRNA levels of alpha 1I and alpha 2I chains might reflect either a transcriptional defect or a decreased stability of mRNA transcripts. We suggest that the association of both pathological chondrocytes producing altered collagen type II and decreased synthesis of type I could be responsible for this peculiar phenotype. The overmodification of alpha 1II CNBr peptides is consistent with the presence of a single-base substitution in the COL2A1 gene. Whether there is a direct causal relationship between the type II collagen defect and the underexpression of type I collagen will require clarification.

A new lethal brittle bone syndrome with increased amount of type V collagen in a patient.

A new lethal brittle bone disease is described in three patients with slender long bones, thin ribs, hypomineralized calvaria, and normal facial appearance. In spite of several limb fractures this syndrome can be differentiated from the lethal forms of osteogenesis imperfecta and is better related to the thin-bone group of lethal dysplasias. Biochemical investigation of collagen from one of the patients by the use of gel electrophoresis and high-pressure liquid chromatography analyses failed to demonstrate any evident defect in the structure of type I collagen chains. Nevertheless collagen extractability from the dermis was altered owing to an increase in the proportion of acid-soluble material. Tritium-proline labeling of cultured fibroblasts confirmed the reduction in total collagen synthesis. This was attributed to a lower type I and type III amount whereas type V collagen level was markedly increased in the cell layer. RNA analysis of the three collagen types with the appropriate cDNA probes confirmed the protein data. Electron microscopic examination of bone and skin showed morphologically abnormal fibroblasts and osteoblasts with an abundant distended rough endoplasmic reticulum and an altered plasma membrane. Unexpected thin fibrils with a banding pattern and surrounding the type I fibrils were observed. They might represent type V collagen. We suggest that, in this patient, the moderate decrease in type I collagen amount is insufficient to account for the radiological findings and that type V collagen overproduction could play a role in the bone brittleness by interfering with the process of mineralization.